Serine proteases are a subgroup of carbonyl hydrolase. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. M. (1974), Sci. Amer., 131, 74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: the subtilisins and the mammalian chymotrypsin related and homologous bacterial serine proteases (e.g., trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977), Ann. Rev. Biochem., 46, 331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.
Subtilisin is a serine endoprotease (MW 27,500) which is secreted in large amounts from a wide variety of Bacillus species and other microorganisms. The protein sequence of subtilisin has been determined from at least four different species of Bacillus. Markland, F. S., et al. (1983), Honne-Seyler's Z. Physiol. Chem., 364, 1537-1540. The three-dimensional crystallographic structure of Bacillus amyloliquefaciens subtilisin to 2.5A resolution has also been reported. Wright, C. S., et al. (1969), Nature, 221, 235-242; Drenth, J., et al. (1972), Eur. J. Biochem., 26, 177-181. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972), Biochemistry, 11, 2439-2449), or product complexes (Robertus, J. D., et al. (1976), J. Biol. Chem., 251, 1097-1103), have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin (Philipp, M., et al. (1983), Mol. Cell. Biochem., 51, 5-32; Svendsen, B. (1976), Carlsberg Res. Comm., 41, 237-291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methione at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965), J. Biol Chem., 244, 5333-5338) and the side chain of serine at residue 221 converted to cysteine by chemical modification (Polgar, et al. (1981), Biochimica et Biophysica Acta. 667, 351-354.)
U.S. Pat. No. 4,760,025 and EPO Publication No. 0 130 756 published Jan. 9, 1985 each disclose the modification of subtilisin amino acid residues corresponding to positions in Bacillus amyloliquefaciens subtilisin tyrosine -1, aspartate +32, asparagine +155, tyrosine +104, methionine +222, glycine +166, histidine 64, glycine +169, phenylalanine +189, serine +33, serine +221, tyrosine +217, glutamate +156 and alanine +152. EPO Publication No. 0 251 446 published Jan. 7, 1988 discloses other amino acid residues in Bacillus amyloliquefaciens subtilisin and their equivalents which may be modified by way of substitution, insertion or deletion and which may be combined with modifications to the residues identified in U.S. Pat. No. 4,760,025 to form useful subtilisin mutants. The particular residues identified herein, however, are not identified in these references.
Similarly, PCT Publication No. W0 89/09819 and WO 89/09830 each published Oct. 19, 1989, disclose subtilisin enzymes made by mutating a nucleotide sequence coding for a subtilisin. Numerous amino acid residues are identified in each of these publications which may be so modified. However, as with the previously identified references, neither identifies the residues of the present invention.
Accordingly, it is an object herein to provide carbonyl hydrolase mutants containing the substitution of amino acid residues in a precursor carbonyl hydrolase corresponding to positions +123 and/or +274 in Bacillus amyloliquefaciens subtilisin. Such mutants generally have at least one property which is different from the same property of the carbonyl hydrolase precursor from which the amino acid of said mutant is derived.
It is further object to provide DNA sequences encoding such carbonyl hydrolase mutants as well as expression vectors containing such mutant DNA sequences.
Still further, another object of the invention is to provide host cells transformed with such vectors as well as host cells which are capable of expressing such DNA to produce carbonyl hydrolase mutants either intracellularly or extracellularly.
The references discussed above are provided solely for their disclosure prior to the filing date of the instant case, and nothing herein is to be construed as an admission that the inventors are not entitled to antidate such disclosure by virtue of a prior invention or priority based on earlier filed applications.